DNA encoding a growth factor specific for epithelial cells

ABSTRACT

Discoveries are disclosed that show particular aspects of recombinant DNA technology can be used successfully to produce hitherto unknown human keratinocyte growth factor (KGF) protein free of other polypeptides. These proteins can be produced in various functional forms from spontaneously secreting cells or from DNA segments .introduced into cells. These forms variously enable biochemical and functional studies of this novel protein as well as production of antibodies. Means are described for determining the level of expression of genes for the KGF protein, for example, by measuring mRNA levels in cells or by measuring antigen secreted in extracellular or body fluids.

This application is a continuation of application Ser. No. 08/106,775,filed Aug. 16, 1993, now abandoned, which is a continuation of Ser. No.07/780,847, filed Oct. 23, 1991, now abandoned, which is a continuationof Ser. No. 07/304,281, filed Jan. 31, 1989, now abandoned.

FIELD OF THE INVENTION

The present invention relates to growth factors, particularly toisolation of a polypeptide growth factor similar to a family of factorsincluding known fibroblast growth factors (FGFs). This invention alsorelates to construction of complementary DNA (cDNA) segments frommessenger RNA (mRNA) encoding the novel growth factor. Further, thisinvention pertains to synthesis of products of such DNA segments byrecombinant cells, and to the manufacture and use of certain other novelproducts enabled by the identification and cloning of DNAs encoding thisgrowth factor.

    ______________________________________                                        ABBREVIATIONS USED IN THIS APPLICATION                                        aFGF         acidic fibroblast growth factor                                  bFGF         basic fibroblast growth factor                                   EGF          epidermal growth factor                                          HSAC         heparin-Sepharose affinity chromatography                        kb           kilobases                                                        kDa          kilodaltons                                                      KGF          keratinocyte growth factor                                       NaDodSO.sub.4 /PAGE                                                                        Sodium dodecylsulphate (SDS)/polyacryl-                                       amide gel electrophoresis                                        RP-HPLC      reversed-phase high performance liquid                                        chromatography                                                   TGFα   transforming growth factor α                               ______________________________________                                    

BACKGROUND OF THE INVENTION

Growth factors are important mediators of intercellular communication.These potent molecules are generally released by one cell type and actto influence proliferation of other cell types (James, R. and Bradshaw,R. A. (1984), Ann. Rev. Biochem. 53, 259-292). Interest in growthfactors has been heightened by evidence of their potential involvementin neoplasia (Sporn, M. B. and Todaro, G. J. (1980), N. Eng. J. Med.303, 878-880). The v-sis transforming gene of simian sarcoma virusencodes a protein that is homologous to the B chain of platelet-derivedgrowth factor (James, R. and Bradshaw, R. A. (1984) Ann. Rev. Biochem.53, 259-292; Doolittle, R. F., et al. (1983) Science 221, 275-277).Moreover, a number of oncogenes are homologues of genes encoding growthfactor receptors (James, R. and Bradshaw, R. A. (1984) Ann. Rev.Biochem. 53, 259-292). Thus, increased understanding of growth factorsand their receptor-mediated signal transduction pathways is likely toprovide insights into mechanisms of both normal and malignant cellgrowth.

One known family of growth factors affecting connective tissue cellsincludes acidic fibroblast growth factor (aFGF), basic fibroblast growthfactor (bFGF), and the related products of the hst, and int-2 oncogenes.

Further, it is known that some growth factors, including the following,have heparin-binding properties: aFGF (Maciag, T., Mehlman, T., Friesel,R. and Schreiber, A. B. (1984) Science 225, 932-935; Conn, G. andHatcher, V. B. (1984) Biochem. Biophys. Res. Comm. 124, 262-268); bFGF(Gospodarowicz, D., Cheng, J., Lui, G.-M., Baird, A. and Bohlen, P.(1984) Proc. Natl. Acad. Sci. USA 81, 6963-6967; Maciag, T., Mehlman,T., Friesel, R. and Schreiber, A. B. (1984) Science 225, 932-935);granulocyte/macrophage colony stimulating factor (James, R. andBradshaw, R. A. (1984) Ann. Rev. Biochem. 53, 259-292); and interleukin3 (James, R. and Bradshaw, R. A. (1984) Ann. Rev. Biochem. 53, 259-292).Each of these polypeptide factors is produced by stromal cells (James,R. and Bradshaw, R. A. (1984) Ann. Rev. Biochem. 53, 259-292, Doolittle,R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C.,Aaronson, S. A. and Antoniades, M. N. (1983) Science 221, 275-277,Roberts, R., Gallagher, J., Spooncer, E., Allen, T. D., Bloomfield, F.and Dexter, T. M. (1988) Nature 332, 376-378). Such factors appear to bedeposited in the extracellular matrix, or on proteoglycans coating thestromal cell surface (James, R. and Bradshaw, R. A. (1984) Ann. Rev.Biochem. 53, 259-292, Roberts, R., Gallagher, J., Spooncer, E., Allen,T. D., Bloomfield, F. and Dexter, T. M. (1988) Nature 332, 376-378). Ithas been postulated that their storage, release and contact withspecific target cells are regulated by this interaction (Roberts, R.,Gallagher, J., Spooncer, E., Allen, T. D., Bloomfield, F. and Dexter, T.M. (1988) Nature 332, 376-378, Vlodavsky, I., Folkman, J., Sullivan, R.,Fridman, R., Ishai-Michaeli, R., Sasse, J. and Klagsburn, M. (1987)Proc. Natl. Acad. Sci. USA 84, 2292-2296).

It is widely recognized, however, that the vast majority of humanmalignancies are derived from epithelial tissues (Wright, N. andAllison, M. (1984) The Biology of Epithelial Cell Populations (OxfordUniversity Press, New York) Vol. 1, pp. 3-5). Effectors of epithelialcell proliferation derived from mesenchymal tissues have been described(James, R. and Bradshaw, R. A. (1984) Ann. Rev. Biochem. 53, 259-292,Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G.,Robbins, K. C., Aaronson, S. A. and Antoniades, M. N. (1983) Science221, 275-2772, Waterfield, M. D., Scrace, G. J., Whittle, N., Strooband,P., Johnson, A., Wasteton, A., Westermark, B., Heldin, C.-H., Huang, J.S. and Deuel, T. F. (1983) Nature 304, 35-39), however, their molecularidentities and structures have not been elucidated.

In light of this dearth of knowledge about such mesenchymal growthfactors affecting epithelial cells, it is apparent that there has been aneed for methods and compositions and bioassays which would provide animproved knowledge and analysis of mechanisms of regulation ofepithelial cell proliferation, and, ultimately, a need for noveldiagnostics and therapies based on the factors involved therein.

This invention contemplates the application of methods of proteinisolation and recombinant DNA technologies to fulfill such needs and todevelop means for producing protein factors of mesenchymal origin, whichappear to be related to epithelial cell proliferation processes andwhich could not be produced otherwise. This invention also contemplatesthe application of the molecular mechanisms of these factors related toepithelial cell growth processes.

SUMMARY OF THE INVENTION

The present invention relates to developments of protein isolation andrecombinant DNA technologies, which include production of novel growthfactor proteins affecting epithelial cells, free of other peptidefactors. Novel DNA segments and bioassay methods are also included.

The present invention in particular relates to a novel protein havingstructural and/or functional characteristics of a known family of growthfactors which includes acidic fibroblast growth factor (aFGF), basicfibroblast growth factor (bFGF) and the related products of the hst, andint-2 oncogenes. This new member of the FGF polypeptide family retainsthe heparin-binding properties of the FGFs but has evolved a uniquetarget cell specificity. This growth factor appears to be specific forepithelial cells and is particularly active on keratinocytes. Therefore,this novel factor has been designated "keratinocyte growth factor"(KGF). Notwithstanding its lack of activity on fibroblasts, since it isthe sixth known member of the FGF polypeptide family, KGF may also bereferred to as FGF-6.

Accordingly, this invention relates, in part, to purified KGF orKGF-like proteins and methods for preparing these proteins. Suchpurified factors may be made by cultivation of human cells whichnaturally secrete these proteins and application of isolation methodsaccording to the practice of this invention. These proteins can be usedfor biochemical and biological studies leading, for example, toisolation of DNA segments encoding KGF or KGF-like polypeptides.

The present invention also relates to such DNA segments which encode KGFor KGF-like proteins. In a principal embodiment, the present inventionrelates to DNA segments, which encode KGF-related products, consistingof: human CDNA clones 32 or 49, derived from polyadenylated RNAextracted from the human embryonic lung fibroblast cell line M426;recombinants and mutants of these clones; and related DNA segments whichcan be detected by hybridization to any of the above human DNA segments,which related segments encode KGF-like proteins or portions thereof.

In the practice of one embodiment of this invention, the DNA segments ofthe invention are capable of being expressed in suitable host cells,thereby producing KGF or KGF-like proteins. The invention also relatesto mRNAs produced as the result of transcription of the sense strands ofthe DNA segments of this invention.

In another embodiment, the invention relates to a recombinant DNAmolecule comprising a vector and a DNA of the present invention. Theserecombinant molecules are exemplified by molecules comprising a KGF cDNAand any of the following vector DNAs: a bacteriophage λ cloning vector(exemplified by λpCEV9); a DNA sequencing plasmid vector (e.g., a pUCvariant); a bacterial gene expression vector (e.g., pKK233-2); or amammalian gene expression vector (such as pMMT).

In still another embodiment, the invention comprises a cell, preferablya mammalian cell, transformed with a DNA of the invention. Further, theinvention comprises cells, including insect cells, yeast cells andbacterial cells such as those of Escherichia coli and B. subtilis,transformed with DNAs of the invention. According to another embodimentof this aspect of the invention, the transforming DNA is capable ofbeing expressed in the cell, thereby increasing in the cell the amountof KGF or KGF-like protein encoded by this DNA.

The primary KGF translation product predicted from its cDNA sequencecontains an N-terminal hydrophobic region which likely serves as asignal sequence for secretion and which is not present in the mature KGFmolecule. In a most preferred embodiment of the gene expression aspectof the invention, the cell transformed by the DNA of the inventionsecretes the protein encoded by that DNA in the (truncated) form that issecreted by human embryonic lung fibroblast cells.

Still further, this invention contemplates KGF or KGF-like proteinsproduced by expression of a DNA of the invention, or by translation ofan RNA of the invention. Preferably, these proteins will be of thesecreted form (i.e., lacking an apparent signal sequence). These proteinfactors can be used for functional studies, and can be purified foradditional structural and functional analyses, such as qualitative andquantitative receptor binding assays.

Moreover, the ability to produce large quantities of this novel growthfactor by recombinant techniques will allow testing of its clinicalapplicability in situations where specific stimulation of growth ofepithelial cells is of particular importance. Accordingly, thisinvention includes pharmaceutical compositions comprising KGF orKGF-like polypeptides for use in the treatment of such conditions,including, for example, healing of wounds due to burns or stimulation oftransplanted corneal tissue.

According to this embodiment of the invention, the novel KGF-likeproteins will be protein products of "unmodified" DNAs and mRNAs of theinvention, or will be modified or genetically engineered proteinproducts. As a result of engineered mutations in the DNA sequencesmodified KGF-like proteins will have one or more differences in aminoacid sequence from the corresponding naturally occurring "wild-type"proteins. According to one embodiment of this aspect of this invention,the modified KGF-like proteins will include "chimeric" moleculescomprising segments of amino acid sequences of KGF and at least oneother member of the FGF peptide family.

Ultimately, given results of analogous successful approaches with otherpeptide factors having similar properties, development of such chimericKGF-like polypeptides should lead to superior, "second generation" formsof KGF-like peptides for clinical purposes. These modified KGF-likeproducts might be smaller, more stable, more potent, and/or easier orless expensive to produce, for example.

This invention further comprises novel bioassay methods for determiningexpression in human cells of the mRNAs and proteins produced from thegenes related to DNA segments of the invention. According to one suchembodiment, DNAs of this invention may be used as probes to determinesteady state levels or kinetics of induction of related mRNAs. Theavailability of the KGF-related cDNA clones makes it possible todetermine whether abnormal expression of this growth factor is involvedin clinical conditions characterized by excessive epithelial cellgrowth, including dysplasia and neoplasia (e.g., psoriasis or malignantor benign epithelial tumors).

This invention also contemplates novel antibodies made against a peptideencoded by a DNA segment of the invention. In this embodiment of theinvention, the antibodies are monoclonal or polyclonal in origin, andare generated using KGF-related polypeptides from natural, recombinantor synthetic chemistry sources.

The antibodies of this invention bind specifically to KGF or a KGF-likeprotein which includes the sequence of such peptide, preferably whenthat protein is in its native (biologically active) conformation. Theseantibodies can be used for detection or purification of the KGF orKGF-like protein factors. In a most preferred embodiment of this aspectof the invention, the antibodies will neutralize the growth promotingactivity of KGF, thereby enabling mechanistic studies and, ultimately,therapy for clinical conditions involving excessive levels of KGF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts results of Heparin-Sepharose affinity chromatography ofconditioned medium from M426 human embryonic fibroblasts. Approximately150 ml of ultrafiltration retentate derived from five liters of M426conditioned medium were loaded onto a heparin-Sepharose column (6 ml bedvolume) in 1 hr. After washing the column with 150 ml of theequilibration buffer, 20 mM Tris-HCl, pH 7.50/0.3M NaCl, the retainedprotein (<5% of the total protein in the retentate) was eluted with amodified linear gradient of increasing NaCl concentration. Fraction sizewas 3.8 ml and flow rate during gradient elution was 108 ml/hr. Two μlof the indicated fractions were transferred to microtiter wellscontaining a final volume of 0.2 ml for assay of ³ H-thymidineincorporation in BALB/MK cells as described in the Methods.

FIGS. 2A, 2B, and 2C illustrates results of further purification of themitogen from human fibroblasts using HPLC with an adsorptive matrix.Panel (A) shows the profile on (A) Reversed-phase C₄ HPLC of BALB/MKmitogenic activity. Active fractions eluted from heparin-Sepharose with0.6M NaCl were processed with the Centricon -10 and loaded directly ontoa C₄ Vydac column (4.6×250 mm) which had been equilibrated in 0.1%trifluoroacetic acid/20% acetonitrile (ACN). After washing the columnwith 4 ml of equilibration buffer, the sample was eluted with a modifiedlinear gradient of increasing % ACN. Fraction size was 0.2 ml and flowrate was 0.5 ml/min. Aliquots for the assay of ³ H-thymidineincorporation in BALB/MK cells were promptly diluted 10-fold with 50μg/ml bovine serum albumin/20 mM Tris-HCl, pH 7.5, and tested at a finaldilution of 200-fold. (B) NaDodSO₄ /PAGE analysis of selected fractionsfrom the C₄ chromatography shown in-panel A. Half of each fraction wasdried, redissolved in NaDodSO₄ /2 mercaptoethanol, heat denatured andelectrophoresed in a 14% polyacrylamide gel which was subsequentlystained with silver. The position of each molecular weight marker (massin kDa) is indicated by an arrow. (C) DNA synthesis in BALB/MK cellstriggered by the fractions analyzed in Panel B. Activity is expressed asthe fold stimulation over background which was 100 cpm.

FIG. 3 presents an alternative purification step to RP-HPLC, usingMolecular sieving HPLC (TSK 3000SW) chromatography of the BALB/MKmitogenic activity. Approximately 50 μl of a Centricon-processed, 0.6MNaCl pool from HSAC were loaded onto a LKB GlasPac TSK G3000SW column(8×300 mm), previously equilibrated in 20 mM Tris-HCl, pH 6.8/0.5M NaCl,and eluted as 0.2 ml fractions at a flow rate of 0.4 ml/min. Aliquots of2 μl were transferred to microtiter wells containing a final volume of0.2 ml for assay of ³ H-thymidine incorporation in BALB/MK cells. Theelution positions of molecular weight markers (mass in kDa) were asindicated by the arrows.

FIG. 4 illustrates a Comparison of BALB/MK DNA synthesis in response toTSK-purified mitogen and other growth factors. Incorporation of ³H-thymidine into trichloracetic acid-insoluble DNA, expressed as foldstimulation over background, was measured as a function of theconcentration of the indicated growth factors. Background values with nosample added were 150 cpm. The results represent mean values of twoindependent experiments. Replicates in each experiment were within 10%of mean values. TSK-purified mitogen, •₋₋₋₋₋₋₋₋₋₋ •; EGF, Δ₋₋₋₋₋₋₋₋₋₋ Δ;aFGF, □₋₋₋₋₋₋₋₋₋₋ □; bFGF, ◯₋₋₋₋₋₋₋₋₋₋ ◯.

FIG. 5 shows Comparative growth of BALB/MK cells in a chemically definedmedium in response to different combinations of growth factors. Cultureswere plated at a density of 2.5×10⁴ cells per dish on 35 mm Petri dishesprecoated with poly-D-lysine/fibronectin in a 1:1 mixture of Eagle'sminimal essential medium and Ham's F12 medium supplemented withtransferrin, Na₂ SeO₃, ethanolamine and the growth factors indicatedbelow. After 10 days, the plates were fixed and stained with Giemsa.Key: a) no growth factor; b) EGF alone; c) insulin alone; d) KGF alone;e) EGF and dialyzed fetal calf serum (final concentration, 10%); f) KGFand EGF; g) KGF and insulin; h) EGF and insulin. Final concentrations ofthe growth factors were as follows: EGF, 20 ng/ml; insulin, 10 μg/ml;and KGF, 40 ng/ml.

FIG. 6 outlines a schematic representation of human KGF cDNA clones.Overlapping pCEV9 clones 32 and 49, used in sequence determination, areshown above a diagram of the complete structure in which untranslatedregions are depicted by a line and the coding sequence is boxed. Thehatched region denotes sequences of the signal peptide. Selectedrestriction sites are indicated.

FIG. 7 documents the KGF cDNA nucleotide and predicted amino acidsequences. Nucleotides are numbered on the left; amino acids arenumbered throughout. The N-terminal peptide sequence derived frompurified KGF is underlined. The hydrophobic N-terminal domain isitalicized. The potential asparagine-linked glycosylation site isoverlined. The variant polyadenylation signals, AATTAA and AATACA, closeto the 3' end of the RNA, are boxed.

FIG. 8 shows identification of KGF mRNAs by Northern blot analysis.Lanes a and c, poly(A)-selected M426 RNA; lanes b and d, total cellularM426 RNA. Filters were hybridized with a ³² P-labeled 695 bp BamHI/BclIfragment from clone 32 (Probe A, FIG. 6), lanes a and b, or a 541 bpApaI/EcoRI fragment from clone 49 (Probe B, FIG. 6), lanes c and d.

FIG. 9 illustrates the topological comparison of the FGF family ofrelated molecules, including KGF, with emphasis on the two proteindomains that share high homology (shaded boxes), the putative signalpeptide sequences (hatched boxes), and the two conserved cysteineresidues (positions labeled with a "C").

FIG. 10 shows Northern blot analysis of KGF mRNA in normal human celllines and tissues, and comparison with mRNA expression of other growthfactors with known activity on epithelial cells. Total cellular RNAswere isolated by cesium trifluoro-acetate gradient centrifugation. 10 μgof RNA were denatured and electrophoresed in 1% formaldehyde gels.Following milk alkali denaturation (50 mM NaOH for 30'), RNA wastransferred to nitrocellulose filters using 1M ammonium acetate as aconvectant. Filters were hybridized to a ³² P-labelled cDNA probecontaining the BamHI/BclI fragment containing the majority of the KGFcoding sequence (A) or similar probes from the other growth factor DNAs.The following human cell types were used: squamous cell carcinomas(A253, A388 and A431); mammary epithelial cells B5/589; immortalizedbronchial epithelial cells (S6 and R1); keratinocytes immortalized withAd12-SV40; primary human keratinocytes; neonatal foreskin fibroblasts,(AG1523) adult skin fibroblasts (501T); and embryonic lung fibroblasts(WI-38 and M426), revealing that a single 2.4 kb transcript was presentin RNA from human embryonic lung fibroblasts and from adult skinfibroblasts, while no transcript was detected in the (B5/589) epithelialor (HA 83) glial cell lines or in primary cultures of human saphenousvein endothelial cells.

DESCRIPTION OF SPECIFIC EMBODIMENTS

This invention relates, in part, to purified KGF or KGF-like proteinsand methods for preparing these proteins. A principal embodiment of thisaspect of this invention relates to homogeneous KGF characterized by anapparent molecular weight of about 28 kDa based on migration in NaDodSO₄/PAGE, movement as a single peak on reversed-phase high performanceliquid chromatography, and a specific activity of at least about 3.4×10⁴units per milligram, and preferably at least about 3.2×10⁵ units permilligram, where one unit of activity is defined as that amount whichcauses half of the maximal possible stimulation of DNA synthesis incertain epithelial (keratinocyte) cells under standard assay conditionsoutlined below.

To identify novel growth factors specific for epithelial cell types, aclonal BALB/c mouse keratinocyte cell line, designated BALB/MK(Weissman, B. E. and Aaronson, S. A. (1983) Cell 32, 599-606) wasemployed as an indicator cell to detect such factors. These cells aredependent for their growth upon an exogenous source of an epithelialcell mitogen even in medium containing serum (Weissman, B. E. andAaronson, S. A. (1983) Cell 32, 599-606). The development of chemicallydefined medium for these cells has made it possible to demonstrate thattwo major mitogenic pathways are required for BALB/MK proliferation. Oneinvolves insulin-like growth factor I (or insulin at high concentration)and the other is satisfied by epidermal growth factor (EGF),transforming growth factor α (TGFα), acidic fibroblast growth factor(aFGF) or basic fibroblast growth factor (bFGF) (Falco, J. P., Taylor,W. G., DiFiore, P. P., Weissman, B. E., and Aaronson, S. A. (1988)Oncogene 2, 573-578).

By using BALB/MK as the prototypical epithelial cell line and NIH/3T3 asits fibroblast counterpart, conditioned media from various human celllines were assayed for new epithelial cell-specific mitogens. Thesebioassays of this invention enabled the purification to homogeneity ofone such novel growth factor, released by a human embryonic lungfibroblast line, and designated herein as keratinocyte growth factor(KGF).

In brief, the bioassay for KGF-like activity under standard conditionscomprises the following steps:

(i) Mouse keratinocytes (BALB/MK cells) are grown in culture toconfluency and then maintained for 24-72 hr in serum-free medium;

(ii) Following addition of test samples, stimulation of DNA synthesis isdetermined by incorporation of ³ H-thymidine into acid-precipitable DNA.

To determine the target cell specificity of a mitogenic growth factor,the DNA synthesis stimulation, expressed as ratio of stimulatedsynthesis over background incorporation of thymidine in the absence ofadded test sample, can be compared to analogous stimulation observed incells other than keratinocytes under the same assay conditions. In suchcomparisons, KGF mitogenic activity will exhibit marked specificity forthe keratinocytes as opposed to fibroblasts (at least about 500-foldgreater stimulation) and lesser but significant (at least about 50-fold)greater activity on keratinocytes than on other exemplary epithelialcell types (see Table 2 for further data, and Materials and Methods inExperimental Section I for details of the standard conditions of thebioassay).

By employing a method of KGF production involving culturing cells andisolating mitogenic activity, which method comprises ultrafiltration,heparin-Sepharose affinity chromatography (HSAC) and adsorptivereversed-phase high performance liquid chromatography (RP-HPLC) or,alternatively, molecular sieving HPLC (TSK-HPLC), according to thepresent invention, a quantity was isolated sufficient to permit detailedcharacterization of the physical and biological properties of thismolecule.

To summarize, the method for production of KGF from producing cells suchas M426 human embryonic fibroblasts (Aaronson, S. A. and Todaro, G. J.(1968) Virology 36, 254-261), for example, comprises the followingsteps:

(i) Preparation of conditioned media (e.g., 10 liters) using monolayercultures cycled from serum-containing to serum-free medium and storingthe serum-free harvest at -70° C. until further use;

(ii) Concentration by ultrafiltration using membranes having a 10 kDamolecular weight cutoff in several successive steps with interveningdilution in buffer (to facilitate removal of low molecular weightmaterials), followed by optional storage at -70° C.;

(iii) Affinity chromatography on heparin attached to a polymeric support(e.g., Sepharose) with elution by a gradient of increasing NaClconcentration;

(iv) Concentration by a factor of at least ten- to twenty-fold withsmall scale ultrafiltration devices with a 10 kDa molecular weightcutoff (e.g., a Centricon-10 microconcentrator from Amicon) and storageat -70° C.

The next step of the purification process comprises either step (v) or,alternatively, step (vi), as follows:

(V) Reversed-phase HPLC of active fractions (0.6M NaCl pool) from theprevious HSAC step in organic solvent systems; or,

(vi) Molecular sieve HPLC (e.g, on a TSK-G3000SW Glas-Pac Column fromLKB) in aqueous buffer at near physiological pH (e.g., Tris-HCl, pH6.8/0.5M NaCl) followed by storage at -70° C.

A preparation made by the TSK step (vi) was almost as pure as oneobtained from RP-HPLC, as judged by silver-stained NaDodSO₄,/PAGE (datanot shown); but the TSK approach provided a far better recovery ofactivity (Table 1). Further, the TSK-purified material had a higherspecific activity than the RP-HPLC material. KGF prepared by the TSKprocedure above stimulated DNA synthesis in epithelial cells atsub-nanomolar concentrations, but failed to induce any thymidineincorporation into DNA of fibroblasts or endothelial cells at comparableor higher concentrations (up to 5 nM). The activity was sensitive toacid, heat and solvents used in the RP-HPLC step. (See ExperimentalSection I for data on sensitivities and further details of theproduction method.)

Using standard methodology well known in the art, an unambiguous aminoacid sequence was determined for positions 2-13 from the amino terminusof the purified KGP, as follows:Asn-Asp-Met-Thr-Pro-Glu-Gln-Met-Ala-Thr-Asn-Val (see ExperimentalSection I).

The present invention also includes DNA segments encoding KGF andKGF-like polypeptides. The DNAs of this invention are exemplified byDNAs referred to herein as: human cDNA clones 32 and 49 derived frompolyadenylated RNA extracted from the human embryonic lung fibroblastcell line M426; recombinants and mutants of these clones; and relatedDNA segments which can be detected by hybridization to these DNAsegments.

As described in Experimental Section II, to search for cDNA clonescorresponding to the known portion of the KGF amino acid sequence, twopools of oligonucleotide probes were generated based upon all possiblenucleotide sequences encoding the nine-amino acid sequence,Asn-Asp-Met-Thr-Pro-Glu-Gln-Met-Ala. A cDNA library was constructed in acDNA cloning vector, λpCEV9, using polyadenylated RNA extracted from thehuman embryonic lung fibroblast cell line M426 which was the initialsource of the growth factor. Screening of the library (9×10⁵ plaques)with the ³² P-labelled oligonucleotides identified 88 plaques whichhybridized to both probes.

Of 10 plaque-purified clones that were analyzed, one, designated clone49, had a cDNA insert of 3.5 kb, while the rest had inserts ranging from1.8 kb to 2.1 kb. Analysis of the smaller clones revealed several commonrestriction sites, and sequencing of a representative smaller clone,designated clone 32, along with clone 49, demonstrated that they wereoverlapping cDNAs (FIG. 6). Alignment of the two cDNAs established acontinuous sequence of 3.85 kb containing the complete KGF codingsequence. The sense strand DNA nucleotide sequence, and the predictedprimary protein sequence encoded, are shown for the full-lengthcomposite KGF cDNA sequence in FIG. 7.

These DNAs, cDNA clones 32 and 49, as well as recombinant forms of thesesegments comprising the complete KGF coding sequence, are most preferredDNAs of this invention.

From the cDNA sequence, it is apparent that the primary KGF, and hsttranslation products contain hydrophobic N-terminal regions which likelyserve as signal sequences, based on similarity to such sequences in avariety of other proteins. Accordingly, this N-terminal domain is notpresent in the purified mature KGF molecule which is secreted by humanembryonic fibroblasts.

Furthermore, KGF shares with all other members of the FGF family twomajor regions of homology, spanning amino acids 65-156 and 162-189 inthe predicted KGF sequence, which are separated by short, nonhomologousseries of amino acids of various lengths in the different familymembers. The sequence of the purified form of KGF contains five cysteineresidues, two of which are conserved throughout the family of FGFrelated proteins. Five pairs of basic residues occur throughout the KGFsequence. This same pattern has been observed in other FGF familymembers.

It should be obvious to one skilled in the art that, by using the DNAsand RNAs of this invention in hybridization methods (such as Southernblot analyses of genomic human DNAs), especially the most preferred DNAslisted herein above, without undue experimentation, it is possible toscreen genomic or cDNA libraries to find other KGF-like DNAs which fallwithin the scope of this invention. Furthermore, by so using DNAs ofthis invention, genetic markers associated with the KGF gene, such asrestriction fragment length polymorphisms (RFLPs), may be identified andassociated with inherited clinical conditions involving this or othernearby genes.

This invention also includes modified forms of KGF DNAs. According to achief embodiment of this aspect of the invention, such modified DNAsencode KGF-like proteins comprising segments of amino acid sequences ofKGF and at least one other member of the FGF peptide family. Thus, forexample, since there is no significant N-terminal homology between thesecreted form of KGF and analogous positions in other FGF-relatedproteins, polypeptides with novel structural and functional propertiesmay be created by grafting DNA segments encoding the distinct N-terminalsegments of another polypeptide in the FGF family onto a KGF DNA segmentin place of its usual N-terminal sequence.

The polypeptide chimeras produced by such modified DNAs are useful fordetermining whether the KGF NH₂ -terminal domain is sufficient toaccount for its unique target cell specificity. Studies on chimerasshould also provide insights into which domains contribute the differenteffects of heparin on their biologic activities.

Indeed, the utility of this approach has already been confirmed by thesuccessful engineering and expression of a chimeric molecule in whichabout 40 amino acids from the NH₂ - terminus of the secreted form of KGF(beginning with the amino terminal cys residue of the mature KGF form,numbered 32 in FIG. 7, and ending at KGF residue 78, arg) is linked toabout 140 amino acids of the C-terminal core of aFGF (beginning atresidue 39, arg, and continuing to the C-terminal end of the aFGF codingsequence. This chimeric product has a target cell preference forkeratinocytes, like KGF, but lacks susceptibility to heparin, acharacteristic which parallels that of aFGF rather than KGF. This novelKGF-like growth factor may have advantages in clinical applicationswhere administration of an epithelial-specific growth factor isdesirable in the presence of heparin, a commonly used anticoagulant.Further details of the construction of this chimeric molecule and theproperties of the polypeptide are described in Experimental Section II.

Other DNAs of this invention include the following recombinant DNAmolecules comprising a KGF cDNA and any of the following exemplaryvector DNAs: a bacteriophage λ cloning vector (λpCEV9); a DNA sequencingplasmid vector (a pUC variant); a bacterial expression vector(pKK233-2); or a mammalian expression vector (pMMT/neo). Suchrecombinant DNAs are exemplified by constructs described in detail inthe Experimental Sections.

Most preferred recombinant molecules include the following: moleculescomprising the coding sequence for the secreted form of KGF and abacterial expression vector (e.g., pKK233-2) or a cDNA encoding theentire primary translation product (including the NH₂ -terminal signalpeptide) and a mammalian expression vector (exemplified by pMMT) capableof expressing inserted DNAs in mammalian (e.g., NIH/3T3) cells.

Construction of recombinant DNAs containing KGF DNA and a bacterialexpression vector is described in Experimental Section II. In brief, KGFcDNA was expressed to produce polypeptide in E. coli by placing itscoding sequence under control of the hybrid trk promoter in the plasmidexpression vector pKK233-2 (Amman, E. and Brosius, J. (1985) Gene 40,183).

Construction of recombinant DNAs comprising KGF DNA and a mammalianvector capable of expressing inserted DNAs in cultured human or animalcells, can be carried out by standard gene expression technology usingmethods well known in the art for expression of such a relatively simplepolypeptide. One specific embodiment of a recombinant DNA of this aspectof the present invention, involving the mammalian vector pMMT, isdescribed further below in this section under recombinant cells of thisinvention.

DNAs and sense strand RNAs of this invention can be employed, inconjunction with protein production methods of this invention, to makelarge quantities of substantially pure KGF or KGF-like proteins.Substantially pure KGF protein thus produced can be employed, usingwell-known techniques, in diagnostic assays to determine the presence ofreceptors for this protein in various body fluids and tissue samples.

Accordingly, this invention also comprises a cell, preferably abacterial or mammalian cell, transformed with a DNA of the invention,wherein the transforming DNA is capable of being expressed. In apreferred embodiment of this aspect of the invention, the celltransformed by the DNA of the invention produces KGF protein in a fullymitogenic form. Most preferably, these proteins will be of a secretedform (i.e., lacking an apparent signal sequence). These protein factorscan be used for functional studies, and can be purified for additionalbiochemical and functional analyses, such as qualitative andquantitative receptor binding assays.

Recombinant E. coli cells have been constructed in a bacterialexpression vector, pKK233-2, for production of KGF, as detailed inExperimental Section II. In summary, several recombinant bacterialclones were tested for protein production by the usual small scalemethods. All recombinants tested synthesized a protein that wasrecognized by antibodies raised against an amino-terminal KGF peptide(see below). One recombinant was grown up in a one liter culture whichproduced recombinant KGF that efficiently stimulated thymidineincorporation into DNA of BALB/MK keratinocyte cells, but was onlymarginally active on NIH/3T3 fibroblasts. Half-maximal stimulation ofthe BALB/MK cells in the standard keratinocyte bioassay was achievedwith a concentration of between 2 to 5 ng/ml, compared to aconcentration of 10 to 15 ng/ml for KGF purified from M426 cells.

One liter of bacterial cells yielded approximately 50 μg of Mono-Spurified recombinant KGF. It will be apparent to those skilled in theart of gene expression that this initial yield can be improvedsubstantially without undue experimentation by application of a varietyknown recombinant DNA technologies.

Recombinant mammalian (NIH/3T3 mouse) cells have also been constructedusing the entire KGF cDNA coding sequence (including the NH₂ -terminalsignal peptide) and the vector pMMT/neo, which carries mousemetallothionine (MMT) promoter and the selective marker gene forneomycin resistance. The cells are being evaluated for KGF production,particularly for secretion of the mature form (lacking signal peptide)produced by human fibroblasts, using bioassays of the present invention.This same vector and host cell combination has been used successfully toexpress several other similar recombinant polypeptides, including highlevels of Platelet-Derived Growth Factor (PDGF) A and B chains (Sakai,R. K., Scharf, S., Faloona, F., Mullis, K. B., Norn, G. T., Erlich, H.A. and Arnheim, N. (1985) Science 230, 1350-1354). Accordingly, it willbe recognized by those skilled in the art that high yields ofrecombinant KGF can be achieved in this manner, using the aforementionedrecombinant DNAs and transformed cells of this invention.

Ultimately, large-scale production can be used to enable clinicaltesting in conditions requiring specific stimulation of epithelial cellgrowth. Materials and methods for preparing pharmaceutical compositionsfor administration of polypeptides topically (to skin or to the corneaof the eye, for example) or systemically are well known in the art andcan be adapted readily for administration of KGF and KGF-like peptideswithout undue experimentation.

This invention also comprises novel antibodies made against a peptideencoded by a DNA segment of the invention. This embodiment of theinvention is exemplified by several kinds of antibodies which recognizeKGF. These have been prepared using standard methodologies well known inthe art of experimental immunology, as outlined in Experimental SectionII. These antibodies include: monoclonal antibodies raised in miceagainst intact, purified protein from human fibroblasts; polyclonalantibodies raised in rabbits against synthetic peptides with sequencesbased on amino acid sequences predicted from the KGF cDNA sequenceexemplified by a peptide with the sequence of KGF residues 32-45,namely, NDMTPEQMATNVR (using standard one-letter code for amino acidsequences; see FIG. 7)!; polyclonal antibodies raised in rabbits againstboth naturally secreted KGF from human fibroblasts and recombinant KGFproduced in E. coli (see above).

All tested antibodies recognize the recombinant as well as the naturallyoccurring KGF, either in a solid-phase (ELISA) assay and/or in a Westernblot. Some exemplary antibodies, which are preferred antibodies of thisinvention, appear to neutralize mitogenic activity of KGF in the BALB/MKbioassay.

Fragments of antibodies of this invention, such as Fab or F(ab)'fragments, which retain antigen binding activity and can be prepared bymethods well known in the art, also fall within the scope of the presentinvention. Further, this invention comprises pharmaceutical compositionsof the antibodies of this invention, or active fragments thereof, whichcan be prepared using materials and methods for preparing pharmaceuticalcompositions for administration of polypeptides that are well known inthe art and can be adapted readily for administration of KGF andKGF-like peptides without undue experimentation.

These antibodies, and active fragments thereof, can be used, forexample, for detection of KGF in bioassays or for purification of theprotein factors. They may also be used in approaches well known in theart, for isolation of the receptor for KGF, which, as described inExperimental Section II, appears to be distinct from those of all otherknown growth factors.

Those preferred antibodies, and fragments and pharmaceuticalcompositions thereof, which neutralize mitogenic activity of KGF forepithelial cells, as indicated by the BALB/MK assay, for instance, maybe used in the treatment of clinical conditions characterized byexcessive epithelial cell growth, including dysplasia and neoplasia(e.g., psoriasis, or malignant or benign epithelial tumors).

This invention further comprises novel bioassay methods for detectingthe expression of genes related to DNAs of the invention. In someexemplary embodiments, DNAs of this invention were used as probes todetermine steady state levels of related mRNAs. Methods for thesebioassays of the invention, using KGF DNAs, and standard Northernblotting techniques, are described in detail in Experimental Section II.

One skilled in the art will recognize that, without undueexperimentation, such methods may be readily applied to analysis of geneexpression for KGF-like proteins, either in isolated cells or varioustissues. Such bioassays may be useful, for example, for identificationof various classes of tumor cells or genetic defects in the epithelialgrowth processes.

Without further elaboration, it is believed that one of ordinary skillin the art, using the preceding description, and following the methodsof the Experimental Sections below, can utilize the present invention toits fullest extent. The material disclosed in the Experimental Sections,unless otherwise indicated, is disclosed for illustrative purposes andtherefore should not be construed as being limitive in any way of theappended claims.

EXPERIMENTAL SECTION I Identification and Characterization of a NovelGrowth Factor Specific for Epithelial Cells

This section describes experimental work leading to identification of agrowth factor specific for epithelial cells in conditioned medium of ahuman embryonic lung fibroblast cell line. The factor, provisionallytermed keratinocyte growth factor (KGF) because of its predominantactivity on this cell type, was purified to homogeneity by a combinationof ultrafiltration, heparin-Sepharose affinity chromatography andhydrophobic chromatography on a C₄ reversed-phase HPLC column, accordingto methods of this invention. KGF was found to be both acid and heatlabile, and consisted of a single polypeptide chain with an apparentmolecular weight of approximately 28,000 daltons. Purified KGF was apotent mitogen for epithelial cells, capable of stimulating DNAsynthesis in quiescent BALB/MK epidermal keratinocytes by more than500-fold with activity detectable at 0.1 nM and maximal at 1.0 nM. Lackof mitogenic activity on either fibroblasts or endothelial cellsindicated that KGF possessed a target cell specificity distinct from anypreviously characterized growth factor. Microsequencing revealed anamino-terminal sequence containing no significant homology to any knownprotein. The release of this novel growth factor by human embryonicfibroblasts indicates that KGF plays a role in mesenchymal stimulationof normal epithelial cell proliferation.

Methods and Materials

Preparation of Conditioned Media. An early passage of M426 humanembryonic fibroblasts (Aaronson, S. A. and Todaro, G. J. (1968) Virology36, 254-261) was plated onto 175 cm² T-flasks and grown to confluenceover 10-14 days in Dulbeccols modified Eagle's medium (DMEM; GIBCO)supplemented with 10% calf serum (GIBCO). Once confluent, the monolayerswere cycled weekly from serum-containing to serum-free medium, thelatter consisting of DMEM alone. The cells were washed twice with 5 mlof phosphate buffered saline prior to addition of 20 ml of DMEM. After72 hrs, culture fluids were collected and replaced with 35 ml ofserum-containing medium. The conditioned medium was stored at -70° C.until further use.

Ultrafiltration. Approximately ten liters of conditioned medium werethawed, prefiltered through a 0.50 micron filter (Millipore HAWP 142 50)and concentrated to 200 ml using the Pellicon cassette system (MilliporeXX42 00K 60) and a cassette having a 10 kDa molecular weight cutoff(Millipore PTGC 000 05). After concentration, the sample was subjectedto two successive rounds of dilution with one liter of 20 mM Tris-HCl,pH 7.5/0.3M NaCl, each followed by another step of ultrafiltration withthe Pellicon system. Activity recovered in the retentate was eitherimmediately applied to heparin-Sepharose resin or stored at -70° C.

Heparin-Sepharose Affinity Chromatography (HSAC). The retentate fromultrafiltration was loaded onto heparin-Sepharose resin (Pharmacia)which had been equilibrated in 20 mM Tris-HCl, pH 7.5/0.3M NaCl. Theresin was washed extensively until the optical density had returned tobaseline and then subjected to a linear-step gradient of increasing NaClconcentration. After removing aliquots from the fractions for thethymidine incorporation bioassay, selected fractions were concentratedten- to twenty-fold with a Centricon-10 microconcentrator (Amicon) andstored at -70*C.

Reversed-Phase HPLC (RP-HPLC). Active fractions (0.6M NaCl pool) fromthe HSAC were thawed, pooled and further concentrated with theCentricon-10 to a final volume of ≦200 μl. The sample was loaded onto aVydac C₄ HPLC column (The Separations Group, Hesperia, Calif.) which hadbeen equilibrated in 0.1% trifluoroacetic acid (TFA, Fluka)/20%acetonitrile (Baker, HPLC grade) and eluted with a linear gradient ofincreasing acetonitrile. Aliquots for the bioassay were immediatelydiluted in a 10-fold excess of 50 μg/ml BSA (Fraction V, Sigma)/20 mMTris-HCl, pH 7.5. The remainder of the sample was dried in a Speed-Vac(Savant) in preparation for structural analysis.

Molecular Sieve HPLC. Approximately 50 μl of the twice concentratedheparin-Sepharose fractions were loaded onto a TSK-G3000SW Glas-PacColumn (LKB) which had been equilibrated in 20 mM Tris-HCl, pH 6.8/0.5MNaCl. The sample was eluted in this buffer at a flow rate of 0.4 ml/min.After removing aliquots for the bioassay, the fractions were stored at-70*C.

NaDodSO₄ -Polyacrylamide Gel Electrophoresis (NaDodSO₄ /PAGE).Polyacrylamide gels were prepared with NaDodSO₄ according to theprocedure of Laemmli (Laemmli, U.K. (1970) Nature 227, 680-685). Sampleswere boiled for 3 min in the presence of 2.5% 2-mercaptoethanol(vol/vol). The gels were fixed and stained with silver (Merril, C. R.,Goldman, D., Sedman, S. A. and Ebert, M. H. (1981) Science 211,1437-1438) using the reagents and protocol from BioRad. Molecular weightmarkers were from Pharmacia.

DNA Synthesis Stimulation. Ninety-six well microliter plates (Falcon No.3596) were precoated with human fibronectin (Collaborative Research) at1 μg/cm² prior to seeding with BALB/MK cells. Once confluent, the cellswere maintained for 24-72 hr in serum-free medium containing 5 Ag/mltransferrin (Collaborative Research) and 30 nM Na₂ SeO₃ (Baker).Incorporation of ³ H-thymidine (5 μm/ml final concentration, NEN) intoDNA was measured during a 6 hr period beginning at 16 hrs followingaddition of samples. The assay was terminated by washing the cells oncewith ice cold phosphate-buffered saline and twice with 5%trichloroacetic acid. The precipitate was redissolved in 0.25M NaOH,transferred into liquid scintillation fluid (Biofluor, NEN) and counted.

Stimulation of DNA synthesis was monitored as described above forBALB/MK cells on a variety of other cell lines. NIH/3T3 fibroblasts(Jainchill, J. L., Aaronson, S. A. and Todaro, G. J. (1969) J. Virol. 4,549-553) were available from the National Institutes of Health, whileCCL208 Rhesus monkey bronchial epithelial cells (Caputo, J. L., Hay, R.J. and Williams, C. D. (1979) In Vitro 15, 222-223) were obtained fromthe American Type Culture Collection. The B5/589 human mammaryepithelial cell line, prepared as described in (Stampfer, M. R. andBartley, J. C. (1985) Proc. Nail. Acad. Sci. USA 82, 2394-2398), wasobtained from Martha Stampfer (Lawrence Berkeley Laboratory). Themammary cells were grown in RPMI 1640 supplemented with 10% fetal calfserum and 4 ng/ml EGF. When maintained in serum-free conditions, thebasal medium was DMEM. Primary cultures of human saphenous veinendothelial cells were prepared and maintained as described elsewhere(Sharerkin, J. B., Fairchild, K. D., Albus, R. A., Cruess, D. F. andRich, N. M. (1986) J. Surgical Res. 41, 463-472). Epidermal growthfactor and insulin were from Collaborative Research. Acidic FGF and bFGFwere obtained from California Biotechnology, Inc. Recombinant TGFα wasobtained from Genentech, Inc. Media and serum were either from GIBCO,Biofluids, Inc. or the NIH media unit.

Proliferation Assay. Thirty-five mm culture dishes were precoatedsequentially with poly-D-lysine (20 μg/cm²) (Sigma) and humanfibronectin, and then seeded with approximately 2.5×10⁴ BALB/MK cells.The basic medium was a 1:1 mixture of Eagle's low Ca 2+ minimalessential medium and Ham's F-12 medium, supplemented with 5 μg/mltransferrin, 30 nM Na₂ SeO₃ and 0.2 mM ethanolamine (Sigma). Medium waschanged every 2 or 3 days. After 10 days, the cells were fixed informalin (Fisher Scientific Co.) and stained with Giemsa (FisherScientific Co.).

Protein microsequencing. Approximately 4 μg (.sup.˜ 150 pmol) of proteinfrom the active fractions of the C₄ column were redissolved in 50% TFAand loaded onto an Applied Biosystems gas-phase protein sequenator.Twenty rounds of Edman degradation were carried out and identificationsof amino acid derivatives were made with an automated on-line HPLC(Model 120A, Applied Biosystems).

Results

Growth Factor Detection and Isolation. Preliminary screening ofconditioned media from various cell lines indicated that media from somefibroblast lines contained mitogenic activities detectable on bothBALB/MK and NIH/3T3 cells. Whereas boiling destroyed the activity onBALB/MK, mitogenic activity on NIH/3T3 remained intact. Based on theknown heat stability of EGF (Cohen, S. (1962) J. Biol. Chem. 237,1555-1562) and TGFα (DeLarco, J. E. and Todaro, G. J. (1978) Proc. Natl.Acad. Sci. USA 75, 4001-4005), it was reasoned that the BALB/MKmitogenic activity might be due to an agent different from these knownepithelial growth factors.

M426, a human embryonic lung fibroblast line, was selected as the mostproductive source of this activity for purification of the putativegrowth factor(s). Ultrafiltration with the Pellicon system provided aconvenient way of reducing the sample volume to a suitable level forsubsequent chromatography. Various combinations of sieving, ion exchangeand isoelectric focusing chromatography were tried during thedevelopment of a purification scheme, but all resulted in unacceptablylow yields on the other hand, heparin-Sepharose affinity chromatography(HSAC), which has been employed in the purification of other growthfactors (Raines, E. W. and Ross, R. (1982) J. Biol. Chem. 257,5154-5160; Shing, Y., Folkman, J., Sullivan, R., Butterfield, C.,Murray, J. and Klagsburn, M. (1984) Science 223, 1296-1299;Gospodarowicz, D., Cheng, J., Lui, G. -M., Baird, A. and Bohlen, P.(1984) Proc. Natl. Acad. Sci. USA 81, 6963-6967; Maciag, T., Mehlman,T., Friesel, R. and Schreiber, A. B. (1984) Science 225, 932-935; Conn,G. and Hatcher, V. B. (1984) Biochem. Biophys. Res. Comm. 124, 262-268;Lobb, R. R. and Fett, J. W. (1984) Biochemistry 23, 6295-6299), provedto be useful as an early purification step in the present invention.While estimates of recovered specific activity were uncertain at thisstage because of the likely presence of other factors, the apparentyield-of activity was 50-70% with a corresponding enrichment ofapproximately 1000 fold.

As shown in FIG. 1, greater than 90% of the BALB/MK mitogenic activityeluted from the HSAC column with 0.6M NaCl. This peak of activity wasnot associated with any activity on NIH/3T3 cells (data not shown). Amuch smaller peak of BALB/MK mitogenic activity consistently emergedwith 0.8-1.2M NaCl.

Due to the reproducibility of the HSAC pattern, active fractions couldbe identified presumptively on the basis of the gradient and opticaldensity profile. Prompt concentration of 10-20 fold with theCentricon-10 was found to be essential for stability, which could bemaintained subsequently at -70° C. for several months.

Final purification was achieved by RP-HPLC with a C₄ Vydac column, apreparative method suitable for amino acid sequence analysis. While theyield of activity from the C₄ step was usually only a few percent, thisloss could be attributed to the solvents employed. In other experiments,exposure to 0.1% TFA/50% acetonitrile for 1 hr at room temperaturereduced the mitogenic activity of the preparation by 98%. Nonetheless,as shown in FIG. 2A, a single peak of BALB/MK stimulatory activity wasobtained, coinciding with a distinct peak in the optical densityprofile. The peak fractions produced a single band upon NaDodSO₄ /PAGEand silver staining of the gel (FIG. 2B), and the relative mitogenicactivity of each tested fraction (FIG. 2C) correlated well with theintensity of the bands across the activity profile.

An alternative purification step to the HPLC technique described above,using sieving chromatography with a TSK G3000SW GlasPac column run inaqueous solution near physiologic pH, resulted in a major peak ofactivity in the BALB/MK bioassay (FIG. 3). This preparation was almostas pure as the one obtained from RP-HPLC as judged by silver-stainedNaDodSO₄ /PAGE (data not shown) but provided a far better recovery ofactivity (Table 1). The TSK-purified material was used routinely forbiological studies as it had a higher specific activity.

In both types of purified preparations (i.e., purified by HPLC ormolecular sieving), the profile of mitogenic activity was associatedwith a distinct band on NaDodSo₄ /PAGE which appeared to beindistinguishable in the two preparations.

                  TABLE 1                                                         ______________________________________                                        Growth Factor Purification                                                                             Total    Specific                                    Purification                                                                              Protein      activity activity                                    step        (mg)         (units)* (units/mg)                                  ______________________________________                                        Conditioned medium                                                                        1.4 × 10.sup.3a                                                                      2.5 × 10.sup.4                                                                   1.8 × 10.sup.1                        (10 liters)                                                                   Ultrafiltration                                                                           1.3 × 10.sup.3a                                                                      3.2 × 10.sup.4                                                                   2.5 × 10.sup.1                        (retentate)                                                                   HSAC        0.73.sup.b   1.6 × 10.sup.4                                                                   2.2 × 10.sup.4                        0.6 MM NaCl pool                                                              TSK-G3000 SW                                                                              8.4 × 10.sup.-3b                                                                     2.7 × 10.sup.3                                                                   3.2 × 10.sup.5                        C.sub.4 -HPLC                                                                             6.1 × 10.sup.-3b                                                                     2.1 × 10.sup.2                                                                   3.4 × 10.sup.4                        ______________________________________                                         Recoveries were calculated by assuming that all of the                        mitogenic activity in the starting material was due to                        the isolated factor.                                                          *One unit of activity is defined as half of the maximal                       stimulation of thymidine incorporation induced by TSK                         purified factor in the BALB/MK bioassay, in which                             approximately 3 ng of the TSKpurified factor stimulated                       1 unit of activity.                                                           .sup.a Protein was estimated by using the Bradford reagent                    from BioRad.                                                                  .sup.b Protein was estimated by using A.sup.1%.sub.214 = 140.            

Physical and Biological Characterization of the Growth Factor. Thepurified factor had an estimated molecular weight of about 28 kDa basedon NaDodSO₄ /PAGE under reducing (FIGS. 2A-2C) and non-reducingconditions (data not shown). This value was in good agreement with itselution position on two different sizing columns run in solventsexpected to maintain native conformation (TSK-G3000-SW, FIG. 3, andsuperose-12, data not shown). From these data, the mitogen appears toconsist of a single polypeptide chain with a molecular weight of 25-30kDa.

The heat and acid lability of the mitogenic activity were demonstratedusing the BALB/MK mitogenesis bioassay. While activity was unaffected bya 10 min incubation at 50° C., it was reduced by 68% after 10 min at 60°C. and was undetectable after 3 min at 100° C. Exposure to 0.5M aceticacid for 60 min at room temperature resulted in a decline in activity to14% of the control. In comparison, the mitogenic activity of the knowngrowth factor, EGF, was not diminished by any of these treatments.

The dose response curve for the purified growth factor depicted in FIG.4 illustrates that as little as 0.1 nM led to a detectable stimulationof DNA synthesis. Thus, the activity range was comparable to that of theother growth factors analyzed to date. A linear relationship wasobserved in the concentration range 0.1-1.0 nM with maximal stimulationof 600 fold observed at 1.0 nM. The novel factor consistently induced ahigher level of maximal thymidine incorporation than EGF, aFGF, or bFGFin the BALB/MK keratinocytes (FIG. 4).

The distinctive target cell specificity of this factor was demonstratedby comparing its activities on a variety of cell types with those ofother growth factors known to possess epithelial cell mitogenicactivity. As shown in Table 2, the newly isolated factor exhibited astrong mitogenic effect on BALB/MK but also induced demonstrableincorporation of thymidine into DNA of the other epithelial cellstested. In striking contrast, the factor had no detectable mitogeniceffects on mouse (or human, data not shown) fibroblasts or humansaphenous vein endothelial cells.

By comparison, none of the other known growth factors appeared topreferentially stimulate keratinocytes. TGFα and EGF showed potentactivity on fibroblasts, while the FGFs were mitogenic for endothelialcells as well as fibroblasts (Table 2). Because of its specificity ofepithelial cells and the sensitivity of keratinocytes in particular, thenovel mitogen was provisionally designated as keratinocyte growth factor(KGF).

To establish that KGF not only would stimulate DNA synthesis but wouldalso support sustained cell growth, the ability of BALB/MK cells to growin a fully-defined, serum-free medium supplemented with this growthfactor was assessed. As shown in FIG. 5, KGF served as an excellentsubstitute for EGF but not insulin (or insulin-like growth factor I) inthis chemically defined medium. Thus, KGF appears to act through themajor signalling pathway shared by EGF, aFGF and bFGF for proliferationof BALB/MK cells.

                  TABLE 2                                                         ______________________________________                                        Target Cell Specificity of Growth Factors                                     Epithelial           Fibroblast Endothelial                                   Growth                         NIH/  Human saphenous                          Factor                                                                              BALK/MK   BS/589  CCL208 3T3S  vein                                     ______________________________________                                        KGF    500-1000 2-3     5-10   <1    <1                                       EGF   100-200   20-40   10-30  10-20 n.d.                                     TGFa  150-300   n.d.    n.d.   10-20 n.d.                                     aFGF* 300-500   2-3     5-10   50-70 5                                        bFGF  100-200   2-3     2-5    50-70 5                                        ______________________________________                                         Comparison of maximal thymidine incorporation stimulated                      by KGF and other growth factors in a variety of cell                          lines, expressed as fold stimulation over background.                         This data represents a summary of four different                              experiments.                                                                  *Maximal stimulation by aFGF required the presence of                         heparin (Sigma), 20 μg/ml.                                                 n.d. = not determined.                                                   

Microsequencing Reveals a Unique N-terminal Amino Acid Sequence of KGF.To further characterize the growth factor, approximately 150 pmol of C₄-purified material were subjected to amino acid sequence analysis. Asingle sequence was detected with unambiguous assignments made forcycles 2-13, as follows:X-Asn-Asp-Met-Thr-Pro-Glu-Gln-Met-Ala-Thr-Asn-Val. High background noiseprecluded an assignment for the first position which is, therefore,indicated by an X.

A computer search using the FASTP program (Lipman, D. J. and Pearson, R.W. (1985) Science 227, 1435-1441) revealed that the N-terminal aminoacid sequence of KGF showed no significant homology to any protein inthe National Biomedical Research Foundation data bank, thus supportingthe novelty of this epithelial growth factor.

Discussion

The studies described in this Experimental Section identified a humangrowth factor which has a unique specificity for epithelial cells. Byemploying ultrafiltration, HSAC and RP-HPLC or TSK sievingchromatography according to the present invention, a quantity sufficientto permit detailed characterization of the physical and biologicalproperties of this molecule was isolated.

A single silver-stained band corresponding to a molecular weight ofabout 28,000 daltons was detected in the active fractions from RP-HPLC,and the intensity of the band was proportional to the level of mitogenicactivity in these fractions. A band indistinguishable from that obtainedby RP-HPLC was seen in the active fractions from TSK chromatography. Thepurified protein stimulated DNA synthesis in epithelial cells atsubnanomolar concentrations, but failed to induce any thymidineincorporation in fibroblasts or endothelial cells at comparable orhigher concentrations (up to 5 nM). This distinctive target cellspecificity combined with the single novel N-terminal amino acidsequence determined from the purified molecule lead to the conclusionthat KGF represents a new growth factor.

In a chemically defined medium the purified factor was able tocomplement the insulin-like growth factor I/insulin growth requirementof BALB/MK cells and therefore must act through a signal transductionpathway shared with EGF, TGFα and the FGFs. Moreover, the new factor wasmore potent than any of the known epithelial cell mitogens instimulating thymidine incorporation in BALB/MK cells. Preliminaryevidence indicates that this factor is also capable of supportingproliferation of secondary cultures of human keratinocytes (data notshown).

Handling and storage of KGF were problematical during its purification.Besides its inherent lability to acid and heat, it was unstable tolyophilization or dialysis. After HSAC, complete loss of activityoccurred within 24 hr despite the use of carrier proteins, heparin,protease inhibitors, siliconized tubes or storage at either 4° or -20°C. Only concentrating the sample at this stage could preserve itsactivity.

Furthermore, in order to transfer the dried, purified factor it wasnecessary to utilize either strong acid or detergent, consistent with anadsorptive tendency or insolubility. Thus, for preservation of activity,the purified factor was maintained in solution at high concentrations at-70° C. where it remained stable for several months.

The ability of KGF to bind heparin may signify a fundamental property ofthis factor that has a bearing on its function in vivo. Growth factorswith heparin-binding properties include aFGF (Maciag, T., Mehlman, T.,Friesel, R. and Schreiber, A. B. (1984) Science 225, 932-935; Conn, G.and Hatcher, V. B. (1984) Biochem. Biophys. Res. Comm. 124, 262-268;Lobb, R. R. and Fett, J. W. (1984) Biochemistry 23, 6295-6299), bFGF(Gospodarowicz, D., Cheng, J., Lui, G. -M., Baird, A. and Bohlen, P.(1984) Proc. Natl. Acad. Sci. USA 81, 6963-6967, Lobb, R. R. and Fett,J. W. (1984) Biochemistry 23, 6295-6299) granulocyte/macrophage colonystimulating factor (Roberts, R., Gallagher, J., Spooncer, E., Allen, T.D., Bloomfield, F. and Dexter, T. M. (1988) Nature 332, 376-378) andinterleukin 3 (Roberts, R., Gallagher, J., Spooncer, E., Allen, T. D.,Bloomfield, F. and Dexter, T. M. (1988) Nature 332, 376-378). Each ofthese is produced by stromal cells (Roberts, R., Gallagher, J.,Spooncer, E., Allen, T. D., Bloomfield, F. and Dexter, T. M. (1988)Nature 332, 376-378; Libermann, T. A., Friesel, R., Jaye, M., Lyall. R.M., Westermark, B., Drohen, W., Schmidt, A., Maciag, T. andSchlessinger, J. (1987) EMBOJ., 61 1627-1632; Shipley, G. D., Sternfeld,M. D., Coffey, R. J. and Pittelkow, M. R. (1988) J. Cell Biochem. Supp12A, 125, abstr. C420). Such factors appear to be deposited in theextracellular matrix, or on proteoglycans coating the stromal cellsurface (Roberts, R., Gallagher, J., Spooncer, E., Allen, T. D.,Bloomfield, F. and Dexter, T. M. (1988) Nature 332, 376-378, Vlodavsky,I., Folkman, J., Sullivan, R., Fridman, R., Ishai-Michaeli, R., Sasse,J. and Klagsburn, M. (1987) Proc. Natl. Acad. Sci. USA 84, 2292-2296).It has been postulated that their storage, release and contact withspecific target cells are regulated by this interaction (Roberts, R.,Gallagher, J., Spooncer, E., Allen, T. D., Bloomfield, F. and Dexter, T.M. (1988) Nature 332, 376-378, Vlodavsky, I., Folkman, J., Sullivan, R.,Fridman, R., Ishai-Michaeli, R., Sasse, J. and Klagsburn, M. (1987)Proc. Natl. Acad. Sci. USA 84, 2292-2296). While mesenchymal-derivedeffectors of epithelial cell proliferation have also been described(Gilchrest, B. A., Karassik, R. L., Wilkins, L. M., Vrabel, M. A. andMaciag, T. (1983) J. Cell Physiol. 117, 2325-240, Chan, K. Y. andHaschke, R. H. (1983) Exp. Eye Res. 36, 231-246, Stiles, A. D., Smith,B. T. and Post, M. (1986) Exp. Lung Res. 11, 165-177), their identitieshave not been elucidated. Its heparin-binding properties, release byhuman embryonic fibroblast stromal cells, and epithelial cell tropismprovide KGF with all of the properties expected of such a paracrinemediator of normal epithelial cell growth.

The partial amino acid sequence determined for this new growth factorhas enabled molecular cloning of its coding sequence and determinationof its structural relationship to known families of growth factors, asdescribed in Experimental Section II, below.

EXPERIMENTAL SECTION II cDNA Sequence of A Novel Epithelial CellSpecific Growth Factor Defines a New Member of the FGF Family

Work in the previous Experimental Section I identified and purified anovel heparin-binding growth factor, designated keratinocyte growthfactor (KGF), which is particularly active on keratinocytes and appearsto be specific for epithelial cells. This second Experimental Sectiondescribes the isolation and characterization of cDNA clones encodingKGF, using synthetic oligonucleotides, based upon the experimentallydetermined NH₂ -terminal amino acid sequence, as hybridization probes.Nucleotide sequence analysis identified a 582-bp open reading framewhich would code for a 194-amino acid polypeptide that is between 41%and 33% identical to the heparin-binding acidic and basic fibroblastgrowth factors (FGFs), and the related products of the hst, and int-2oncogenes. The KGF gene RNA transcript is expressed in normalfibroblasts of both embryonic and adult origin, but not in epithelial,endothelial or glial cells. Thus, KGF appears to be normally expressedby the mesenchyme, indicating a role in the regulation of epithelialcell proliferation.

Materials and Methods

Isolation of cDNA clones. The purification and N-terminal sequencing ofKGF has been previously described (see Experimental Section I, above andRubin, J. S., Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S. andAaronson, S. A. (1989) Proc. Natl. Acad. Sci. USA (in press), February,1989). Pools (50 pmole) of deoxyoligonucleotides described under Resultswere 5' end-labelled using 83 pmole of τ-³² P-ATP (3000 Ci/mmole,Amersham) and 10 units of T4 polynucleotide kinase. The recombinantphage carrying cDNA clones were replica plated onto nitrocellulosefilters and hybridized with ³² P-labelled deoxyoligonucleotides in 20%formamide, 10% dextran sulphate, 10 mM Tris-HCl (pH 7.5), 8×SSC, 5×Denhardt's and 50 μg/ml denatured salmon sperm DNA, overnight at 42° C.Filters were washed in 0.5×SSC, 0.1% SDS at 50° C. and exposed to KodakX-omat AR film.

DNA sequencing. The nucleotide sequence of the KGF cDNA was determinedby the dideoxy chain termination method (Sanger, F., Nicklen, S. andCoulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467), ofoverlapping restriction fragments, subcloned into pUC vectors(Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene 33, 103-119)

Construction of a bacterial expression vector for KGF cDNA. KGF cDNAencoding the mature, secreted form of the polypeptide was placed undercontrol of the hybrid trk promoter in the plasmid expression vectorpKK233-2 (Amman, E. and Brosius, J. (1985) Gene 40, 183), as follows. Toaccomplish this, a specific length of KGF cDNA that contained theinformation to code for the mature KGF molecule (i.e., without itssignal peptide) was amplified using the polymerase chain reaction (PCR)technique (Sakai, R. K., Scharf, S., Faloona, F., Mullis, K. B., Norn,G. T., Erlich, H. A. and Arnheim, N. (1985) Science 230, 1350-1354). Thefragment was directionally inserted between two sites in the vector,namely the NcoI site, made blunt ended by S1 nuclease digestion, and theHindIII site, using standard recombinant DNA methodology. The ends ofthe KGF cDNA produced by the PCR method were as follows: the 5' end wasblunt and began with an ATG codon, followed by the codon TGC for cysresidue, number 33, which is the amino terminal residue of the matureform of KGF (see FIG. 7), and then the entire KGF coding sequence. Thestop codon, TAA, and the four bases immediately following, TTGC, werealso included on the 3' end of the cDNA. The primer used in the PCRmethod to direct DNA synthesis to the desired position on the 3' end ofthe cDNA included a HindIII site for insertion of the amplified cDNAinto the vector DNA.

Production of antibodies against KGF and KGF-related peptides.Monoclonal antibodies were raised in mice against intact, purifiedprotein from human fibroblasts using 5 or more subcutaneous injections.Test bleeds were screened with a solid-phase (ELISA) assay using highlypurified KGF from human epithelial cells as antigen. Hybridomas wereprepared by routine methods and supernatants were screened with theELISA assay to detect KGF-reactive antibodies. Positive clones wereserially subcloned by the usual methods, and selected subclones weregrown as ascites tumors in mice for production of large amounts ofantibodies. Antibodies were purified from ascites fluids employingstandard techniques hydroxyapatite or immunoaffinity resins).

Polyclonal antibodies against a synthetic peptide were raised in rabbitsby standard methods, as follows. The peptides were made by solid phasetechnology and coupled to thyroglobulin by reaction with glutaraldehyde.Serial subcutaneous injections were made and test bleeds were screenedby ELISA as well as other techniques, including Western blot analysisand mitogenesis bioassay. IgG immunoglobulins were isolated by affinitychromatography using immobilized protein G.

Polyclonal antibodies were raised in rabbits against both naturallysecreted KGF from human fibroblasts and recombinant KGF produced in E.coli (see next section), using the following protocol:

i) Initial injection and first boost were administered in the inguinallymph nodes;

ii) subsequent boosts were made intramuscularly. Screening of testbleeds included ELISA as well as Western blot analysis and mitogenesisbioassay, and IgG was purified as for antibodies against syntheticpeptides, above.

Results

Isolation of cDNA clones encoding the novel growth factor. To search forcDNA clones corresponding to the KGF coding sequence, two pools ofoligonucleotides with lengths of 26 bases were generated based upon anine-amino acid sequence, Asn-Asp-Met-Thr-Pro-Glu-Gln-Met-Ala, asdetermined by microsequencing of purified KGF (see Experimental SectionI, above and reference II-3. One oligonucleotide pool contained amixture of all 256 possible coding sequences for the nine amino acids,while the other contained inosine residues at the degenerate thirdposition of the codons for Thr and Pro.

This latter design reduced the number of possible coding sequences inthe pool to 16. Inosine in a tRNA anticodon can form hydrogen bonds withA, C or U (Crick, F. H. C. (1966) J. Mol. Biol. 19, 548-555), andoligonucleotides that contain deoxyinosine have been shown to hybridizeefficiently with the corresponding cDNA (Ohtsuka, E., Matsuki, S.,Ikehara, M., Takashi, Y. and Matsubara, K. (1985) J. Biol. Chem. 260,2605-2608).

A cDNA library was constructed in a cDNA cloning vector, pCEV9 (Miki,T., Matsui, T., Heidaran, M. and Aaronson, S. A., unpublishedobservations) using polyadenylated RNA extracted from the humanembryonic lung fibroblast cell line M426 (Aaronson, S. A. and Todaro, G.J. 1968, Virology 36, 254-261), the initial source of the growth factor.Screening of the library (9×10⁵ plaques) with the ³² P-labelled 26-meroligonucleotides identified 88 plaques which hybridized to both pools ofoligonucleotide probes.

Characterization and sequencing of selected cDNA clones. Of 10plaque-purified clones that were analyzed, one, designated clone 49, hada cDNA insert of 3.5 kb, while the rest had inserts ranging from 1.8 kbto 2.1 kb.

Analysis of the smaller clones revealed several common restrictionsites. Sequencing of a representative smaller clone, designated clone32, along with clone 49, demonstrated that they were overlapping cDNAs(FIG. 6).

Description of the sequence encoding the KGF polypeptide. Alignment ofthe two cDNAs (clones 32 and 49) established a continuous sequence of3.85 kb containing the complete KGF coding sequence (FIG. 7). An ATGlikely to be an initiation codon was located at nucleotide position 446,establishing a 582-base pair open reading frame that ended at a TAAtermination codon at position 1030. This open reading frame would encodea 194-amino acid polypeptide with a calculated molecular weight of22,512 daltons.

The sequence flanking the ATG codon did not conform to the proposedGCC(G/A) CCATGG consensus for optimal initiation by eukaryotic ribosomes(Kozak, M. (1987) Nucl. Acids Res. 13, 8125-8148), however, there was anA three nucleotides upstream of the ATG codon. An A at this position isthe most highly conserved nucleotide in the consensus. This ATG codonwas preceded 85 nucleotides upstream by a TGA stop codon in the samereading frame.

A 19-amino acid sequence that was homologous to the experimentallydetermined NH₂ -terminus of purified KGF began 32 amino acids downstreamof the proposed initiation codon. There was complete agreement betweenthe predicted and experimentally determined amino acid sequences, whereunambiguous assignments could be made.

To search for homology between KGF and any known protein, a computersearch of the National Biomedical Research Foundation data base usingthe FASTP program of Lipman and Pearson was conducted (Lipman, D. J. andPearson, R. W. (1985) Science 227, 1435-1443). By this approach, astriking degree of relatedness between the predicted primary structureof KGF and those of acidic and basic FGF, as well as the related hst,and int-2-encoded proteins was revealed.

Expression of mRNA transcripts of the KGF gene in human cells. Inpreliminary attempts to examine expression of KGF mRNA in human cells, aprobe spanning the majority of the KGF coding sequence (Probe A, FIG. 6)detected a single 2.4 kb transcript by Northern blot analysis of totalM426 RNA (FIG. 8). This was considerably shorter than the length of thecomposite cDNA sequence, 3.85 kb.

However, on screening poly(A)-selected M426 RNA, an additionaltranscript of approximately 5 kb was detected. Furthermore, a probederived from the untranslated region of clone 49, 3' to the end of clone32 (Probe B, FIG. 6), hybridized only to the larger message (FIG. 8).Thus, it appears that the KGF gene is transcribed as to alternate RNAs.Two other members of the FGF gene family, bFGF (Abraham J. A., et al.(1986) Science 233, 545-548) and int-2 (Mansour, S. L. and Martin, G. R.(1988) EMBO J. 1, 2035-2041), also express multiple RNAs, thesignificance of which remains to be determined.

To investigate the normal functional role of KGF, the expression of itstranscript in a variety of human cell lines and tissues was examined. Asshown in FIG. 10, the predominant 2.4 kb KGF transcript was detected ineach of several stromal fibroblast lines derived from epithelial tissuesof embryonic, neonatal and adult sources, but not from epithelial celllines of normal origin. The transcript was also detected in RNAextracted from normal adult kidneys and organs of the gastrointestinaltract, but not from lung or brain. The striking specificity of KGF RNAexpression in stromal cells from epithelial tissues indicated that thisfactor plays a normal role in mesenchymal stimulation of epithelial cellgrowth.

For comparison, the mRNAs of other growth factors with known activity onepithelial cells were also analyzed in the same tissues as listed above.Among the epithelial and stromal cell lines analyzed, there was noconsistent pattern of expression of aFGF or bFGF transcripts (FIG. 10).The EGF transcript was not expressed in any of the same cell lines, andwas only observed in kidney, among the various tissues. Finally, theTGFα message was not detected in any of the stromal fibroblast lines andwas expressed at varying levels in each of the epithelial cell lines. Itwas also detected at low levels in kidney among the tissues examined(FIG. 10).

Inhibition of KGF mitogenic activity by heparin. Heparin has been shownto substantially increase the mitogenic activity of aFGF for a varietyof target cells in culture, and to stabilize it from heat inactivation(II-21 and II-22).

Despite binding tightly to bFGF, heparin had minimal effects on itsmitogenic activity II-22. In view of the relatedness of KGF to the FGFs,the effect of heparin on KGF mitogenic activity was examined. As shownin Table 3, thymidine incorporation by BALB/MK cells in response to KGFwas inhibited 16 fold when heparin was included in the culture medium.In contrast, the activities of both aFGF and bFGF were increased by thesame treatment.

                  TABLE 3                                                         ______________________________________                                        Effect of Heparin on KGF Mitogen Activity.                                               BALB/MK         NIH/3T3                                            Growth Factor                                                                              -      +          -    +                                         ______________________________________                                         KGF         150    9.5        <1   <1                                        aFGF         106    259        10.4 68                                        bFGF          30    124        45.7 70                                        ______________________________________                                    

Cells were plated in microtiter plates, grown to confluence in serumcontaining media and then placed in a serum-free medium for 24-72 hrprior to sample addition. Mitogenesis assays were performed as described(see Experimental Section I, above II-3. Where indicated, heparin wasincluded in the culture media at a final concentration of 20 μg/ml. Theconcentration of all the growth factors was 50 ng/ml. The resultsrepresent fold stimulation of ³ H-thymidine incorporation in theindicated assay cell in the presence (+) or absence (-) of heparin. Eachvalue represents the mean result from two independent experiments inwhich each point, in turn, represents the mean value of duplicateanalyses.

Production of anti-KGF antibodies. Several kinds of antibodies whichrecognize KGF or KGF-like polypeptides have been prepared using standardmethodologies well known in the art of experimental immunology andsummarized in the Methods section, above. There include: monoclonalantibodies raised in mice against intact, purified protein from humanfibroblasts; polyclonal antibodies raised in rabbits against syntheticpeptides with sequences based on amino acid sequences predicted from theKGF cDNA sequence; polyclonal antibodies raised in rabbits against bothnaturally secreted KGF from human fibroblasts and recombinant KGFproduced in E. coli (see next section).

Monoclonal antibodies from three different hybridomas have beenpurified. All three recognize the recombinant as well as the naturallyoccurring KGF in a solid-phase (ELISA) assay. None cross-reacts with KGFunder denaturing conditions (in a Western blot), and none neutralizesmitogenic activity of KGF in the BALB/MK bioassay.

Polyclonal antibodies were generated with a synthetic peptide with theamino acid sequence NDMTPEQMATNVR, corresponding to residues numbered 32through 44 in KGF (see FIG. 7), plus an R (Arg) residue instead of theactual Ash residue encoded by the cDNA at position 45. The Ash residueis probably glycosylated in the natural KGF polypeptide and, therefore,appeared to be Arg in the amino acid sequencing data obtained directlyfrom that polypeptide (see Discussion, below). Polyclonal antibodiesgenerated with this synthetic peptide recognize both naturally occurringand recombinant KGF in ELISA and Western blot analyses at a level ofsensitivity of at least as low as 10 ng protein. These antibodies,however, do not neutralize mitogenic activity of KGF in the BALB/MKbioassay.

Polyclonal antisera against intact natural KGF protein recognizes KGF inboth ELISA and Western blot assays. Such antibodies also appear toinhibit mitogenic activity of KGF in the BALB/MK bioassay.

Expression of KGF cDNA in E. coli. KGF cDNA was expressed to producepolypeptide, in E. coli by placing its coding sequence under control ofthe hybrid trk promoter (comprising elements of trp and lac promoters),in the plasmid pKK233-2 (Amman, E. and Brosius, J. (1985) Gene 40, 183).To accomplish this, a specific length of KGF cDNA that contained theinformation to code for the mature KGF molecule (i.e., without itssignal peptide) was amplified using the polymerase chain reactiontechnique (Sakai, R. K., Scharf, S., Faloona, F., Mullis, K. B., Norn,G. T., Erlich, H. A. and Arnheim, N. (1985) Science 230, 1350-1354). Thefragment was directionally inserted between two sites in the vector,namely the NcoI site, made blunt ended by S1 nuclease digestion, and theHindIII site, using standard recombinant DNA methodology. Selectedrecombinants were sequenced at their cDNA 5' ends to ensure correctalignment of the ATG initiation codon with the regulatory elements ofthe trk promoter.

Several recombinants were tested for protein production by the usualsmall scale methods. In brief, the clones were grown to mid-exponentialphase (OD₅₉₅ .sup.˜ 0.5), treated with 1 mM isopropylβ-D-thiogalactopyranoside (IPTG) for 90 minutes, and cell extracts wererun on SDS-polyacrylamide gels for Western blot analysis. Allrecombinants tested synthesized a protein that was recognized byantibodies raised against an amino-terminal KGF peptide. One recombinantwas selected which showed the greatest induction from IPTG, for furtherprotein analyses.

One liter of bacteria was grown up in NZY broth containing 50 μg/mlampicillin and 12.5 μg/ml tetracycline, to OD₅₉₅ .sup.˜ 0.5, and treatedfor 90 min. with IPTG. The cells were collected by centrifugation,resuspended in 50 mM sodium phosphate (pH 7.3), 0.2M NaCl, and lysed bysonication. Cell debris was removed by centrifugation, and lysateapplied directly to a heparin-Sepharose affinity column.

As determined by Western blot analysis and mitogenic activity inkeratinocytes, recombinant KGF was eluted in 0.5-0.6M NaCl. Subsequentpurification of the HSAC material with a Mono-S (FPLC) column(Pharmacia) yielded a preparation of KGF estimated to be ≧90% pure, asjudged by electrophoretic analysis using SDS-polyacrylamide gels andsilver-staining.

Recombinant KGF efficiently stimulated thymidine incorporation intoBALB/MK keratinocyte cells, but was only marginally active on NIH/3T3fibroblasts. Half-maximal stimulation of the BALB/MK cells in thestandard keratinocyte bioassay was achieved with a concentration ofbetween 2 to 5 ng/ml, compared to a concentration of 10 to 15 ng/ml forKGF purified from M426 cells. One liter of bacterial cells yieldedapproximately 50 μg of Mono-S purified recombinant KGF.

Construction of a chimera containing KGF and aFGF sequences. The studiesabove indicated that KGF possessed two distinctive characteristics whichmight be encoded by distinct portions or domains of the polypeptidesequence, as is well known to occur in coding sequences of othermultifunctional polypeptides. To test this possibility, a chimeric DNAsegment encoding the NH₂ -terminal sequence of KGF grafted onto theC-terminal core of aFGF was constructed, as follows. A Sau3A restrictionenzyme-site (GATC) in the 5' end of the KGF cDNA, within codons forresidues 76, 77, and 78 (Tyr, Leu, and Arg, respectively; see FIG. 7)was cut and joined to an homologous site in the aFGF cDNA within codonsfor amino acids 39 (Arg) and 40. The 3' and 5' ends of this chimeric DNAwere joined to the vector DNA of the plasmid pKK233-2 by the same methodused for insertion of the KGF cDNA encoding the secreted form ofpolypeptide (see Methods, above).

When recombinant E. coli cells were constructed using the vectorcarrying the chimera, and expression tests were conducted as describedfor mature KGF, above, a novel product with properties of both KGF andaFGF was produced. The peptide was enriched by heparin-Sepharosechromatography and found to have a target cell preference forkeratinocytes, like KGF, with minimal activity on fibroblasts (NIH/3T3).The mitogenic activity of this chimeric polypeptide lacks, however,susceptibility to inhibition by heparin, a characteristic whichparallels that of aFGF rather than KGF. In fact, the mitogenic activityon keratinocytes is actually enhanced by heparin, as is the case foraFGF. Thus the peptide domains responsible for target cell specificityand heparin sensitivity are clearly distinct and readily separable inKGF, according to the practice of the present invention.

Discussion

The experiments described in this section illustrate the practice ofseveral principal embodiments of the present invention. These includeisolation of cDNAs encoding KGF, expression of such cDNAs in recombinantcells, production of various antibodies reactive with KGF, andconstruction and expression of a chimeric cDNA encoding a novel growthfactor with amino acid sequences and related functionalities of both KGFand aFGF. The following points related to these embodiments may also benoted to enhance the understanding of the present invention.

The sequence predicted from the KGF cDNA agreed with the amino acidsequence determined from the purified KGF form secreted by humanfibroblasts. Moreover, the sequence offered potential explanations forpositions where definitive amino acid assignments could not be made bydirect amino acid sequencing. Residues 32 and 46 are predicted from thecDNA sequence to be cysteines, and hydrolyzed derivatives of unmodifiedcysteine residues are not detectable following Edman degradation. Thepredicted KGF amino acid sequence also contained one potential N-linkedglycosylation site (Asn-X-Ser/Thr) from residues 45 through 47. If Asn45 were glycosylated, it would not be detected by the amino acidsequencing methods employed here. In fact, KGF migrates as a broad bandon NaDodSO₄ /PAGE at a higher molecular weight than predicted for thepurified protein. This may be accounted for by glycosylation.

The FGFs are heparin-binding mitogens with broad target cellspecificities (Thomas, K. (1987) FASEB J. 1, 434-440). The hst gene wasidentified as a transforming gene from a human stomach tumor (Taira etal., Proc. Natl. Acad. Sci. USA 84: 2980-2984 (1987), adjacent normalstomack tissue (Yoshida et al., Proc. Natl. Acad. Sci. USA 84:7305-7309(1987), and from Kaposi's sarcoma (Delli-Bovi et al., Proc. Natl. Acad.Sci. USA 84: 5660-5664 (9187), by standard NIH/3T3 transfection assays.The product of the int-2 gene is expressed normally during mouseembryogenesis (Jakobovits, A., Shackleford, G. M., Varmus, H. E. andMartin, G. R. (1986) Proc. Natl. Acad. Sci. USA 83, 7806-7810) andaberrantly after proviral integration of mouse mammary tumor virus(Peters, G., Brookes, S. and Dickson, S. (1983) Cell 33, 364-377). Thehst gene was identified as a transforming gene from a human stomachtumor (II-11), adjacent normal stomach tissue (II-12) and from Kaposi'ssarcoma (II-13), by standard NIH/3T3 transfection assays.

KGF is the sixth member of the fibroblast growth factor family to beidentified (Zhan, X., Bates, B., Hu, X. and Goldfarb, M. (1988) Mol.Cell. Biol. 8, 3487-3495). While the name FGF-6 does not seem suitablebecause KGF is devoid of activity on fibroblasts, this nomenclature mayalso be used for this growth factor, to denote its structuralrelationship to the FGF family. As all previously characterized growthfactors either exclude epithelial cells as targets or include them amonga number of sensitive target cells, the highly specific nature of KGFmitogenic activity for epithelial cells, and the sensitivity ofkeratinocytes in particular, make it unique.

In studies to date, expression of the KGF transcript appears to bespecific for stromal cells derived from epithelial tissues, suggestingits function in normal epithelial cell proliferation. The availabilityof the KGF cDNA clone will make it possible to determine whetherabnormal expression of this growth factor can be implicated in clinicalconditions characterized by epithelial cell dysplasia and/or neoplasia.Moreover, the ability to produce large quantities of this novel growthfactor by recombinant techniques should allow testing of its clinicalapplicability in situations where specific growth of epithelial cells isof particular importance.

Alignment of the KGF sequence with the five other proteins of the FGFfamily revealed two major regions of homology, spanning amino acids65-156 and 162-189 in the predicted KGF sequence, which were separatedby a short, nonhomologous series of amino acids with varying lengths indifferent members of the family (FIG. 9). In the case of int-2, thelength of this sequence was 17 residues, while in hst, the twohomologous regions were contiguous. In KGF the intervening sequenceconsisted of five amino acids.

In the aligned regions, the KGF amino acid sequence was about 44%identical to int-2 (mouse), 41% identical to FGF-5 (human), 39%identical to bFGF (human), 37% identical to aFGF (human) and 33%identical to hst (human). In this same region, all six proteins wereidentical at 19% of the residues, and allowing for conservativesubstitutions, they showed 28% homology.

As shown in FIG. 9, the amino termini of these related proteins arenonhomologous and of variable length. The primary KGF, and hsttranslation products contain hydrophobic N-terminal regions which likelyserve as signal sequences (von Heijne, G. (1986) Nucl. Acids Res. 14,4683-4690). The fact that this N-terminal domain is not present in themature KGF molecule (FIG. 7) further supports this conclusion. Incontrast, the FGFs are synthesized apparently without signal peptides(Thomas, K. (1987) FASEB J. 1, 434-440). The int-2 protein contains anatypically short region of N-terminal hydrophobic residues (von Heijne,G. (1986) Nucl. Acids Res. 14, 4683-4690), but it is not known if theprotein is secreted. Moreover, the int-2 protein contains a long C-terminal extension compared to the other family members.

Purified KGF contains five cysteine residues, two of which are conservedthroughout the family of FGF related proteins (FIG. 9). Also of note arethe five pairs of basic residues throughout the KGF sequence. This samepattern has been observed in other FGF family members and may beinvolved in their interaction with heparin (Schwarzbauer, J. E., Tamkum,J. M., Lemischka, I. R. and Hynes, R. O. (1983) Cell 35, 421-431).Dibasic sites are also common targets for proteolytic processing andsuch processing might account for the microheterogeneity observed insome KGF preparations (unpublished data).

The KGF cDNA sequence was AT rich throughout its length, butparticularly so in the 3' untranslated region where the AT content was70% as compared to 60% in the putative coding sequence and 63% in the 5'untranslated region. The 3' untranslated region contained a large numberof ATTTA sequences, which have been proposed to be involved in theselected degradation of transiently expressed, unstable RNAs (Shaw, G.and Kamen, R. (1986) Cell 46, 659-667). There was no classical AATAAApolyadenylation signal but two variant sequences, AATTAA and AATACA(Birnsteil, M. L., Busslinger, M. and Strub, K. (1985) Cell 41,349-359), were detected 24 and 19 nucleotides, respectively, upstream ofthe poly(A) sequence at the 3' end of the cDNA.

It has been suggested that the heparin effect on acidic FGF is eitherdue to stabilization of the active conformation of the growth factor orto formation of a tertiary complex with acidic FGF and its receptor(Schrieber, A. B., Kenny, J., Kowalski, W., Friesel, J., Mehlman, T. andMaciag, T. (1985) Proc. Natl. Acad. Sci. USA 82, 6138-6142,Gospodarowizc, O. and Cheng, J. (1986) J. Cell Physiol. 128, 475-485).If so, heparin may stabilize a conformation of KGF that is not as activeas the free molecule, or form a tight complex that is unable toefficiently interact with its receptor.

While its ability to bind heparin reflects the structural similaritiesof KGF with the FGF's, the differences in target cell specificitiesbetween these related mitogens is remarkable. The FGF's induce divisionof most nonterminally differentiated cells of both embryonic mesodermaland neuroectodermal origin. In addition to fibroblasts and vascularendothelial tissues, mesodermally derived targets in culture includemyoblasts, chondrocytes and osteoblasts (Thomas, K. A. andGiminez-Gallego, G. (1986) Trends Biochem. Soc. 11, 81-84). FGF's arealso mitogenic for glial astrocytes and neuroblasts (Gensburger, C.,Labourdette, G. and Sensembrenner, M. (1987) FEBS Lett. 217, 1-5). Theproduct of the oncogene isolated from Kaposi's sarcoma, which isidentical to hst, also stimulates proliferation of NIH/3T3 and capillaryendothelial cells (Delli-Bovi, P., Curatola, A. M., Kern, F. G., Greco,A., Ittman, M. and Basilico, C. (1987) Cell 50, 729-737). To date, KGFinduced mitogenesis has only been observed in epithelial cells, and theabsence of any detectable activity in fibroblasts or endothelial cellshas also been demonstrated (see Experimental Section I, above and Rubinet al., Proc. Natl. Acad. Sci. USA 86:802-806 (1989). It seems likely,therefore, that KGF acts through a different cell surface receptor thanthe FGFs.

There is no significant N-terminal homology between KGF and otherFGF-related proteins. Thus, the construction of chimeric moleculesbetween KGF and a prototype FGF was undertaken to determine whether theKGF N-terminal domain is sufficient to account for its unique targetcell specificity. The results on the first such recombinant polypeptidesequence indicate that the N-terminal domain of KGF essentially encodesthe cell preference for keratinocytes, while the susceptibility of KGFto heparin is encoded somewhere in the C-terminal core region which wasreplaced by sequences of aFGF. This novel KGF-like growth factor mayhave advantages in clinical applications where administration of anepithelial-specific growth factor is desirable in the presence ofheparin, a commonly used anticoagulant. Additional studies on chimerasshould also provide insights into which specific domains in theC-terminal core contribute the different effects of heparin on theirbiologic activities.

For purposes of completing the background description and presentdisclosure, each of the published articles, patents and patentapplications heretofore identified in this specification is herebyincorporated by reference into the specification.

The foregoing invention has been described in some detail for purposesof clarity and understanding. It will also be obvious that variouscombinations in form and detail can be made without departing from thescope of the invention.

What is claimed is:
 1. An isolated DNA molecule encoding a keratinocytegrowth factor (KGF) polypeptide having preferential mitogenic activityon cells of epithelial origin, the DNA molecule selected from the groupconsisting of:(a) a cDNA molecule comprising the DNA sequence of FIG.II-1B; (b) a cDNA molecule comprising the polypeptide coding region inFIG. II-1B; (c) a cDNA molecule as defined in (b) further comprising a5' ATG; (d) a human DNA molecule which encodes an mRNA that hybridizesto the 695-bp BamHI/BclI cDNA fragment as set forth in FIGS. II-1A andB, under conditions wherein such BamHI/BclI fragment hybridizes to a 2.4kb KGF mRNA transcript expressed in a M426 cell line, but not to humanaFGF or human bFGF mRNA transcripts in RNA samples from cell lines whichexpress such transcripts; and (e) a DNA molecule which is degeneratefrom and encodes a polypeptide encoded by the DNA molecule defined inone of (a)-(d).
 2. A DNA molecule according to claim 1 lacking thenucleic acid sequence that encodes amino acids 1-31 of FIG. II-1B. 3.The DNA molecule of claim 1, wherein said KGF polypeptide showspreferential mitogenic activity for BALB/MK epithelial cells, but notNIH/3T3 fibroblast cells.
 4. An isolated DNA molecule encoding akeratinocyte growth factor (KGF) polypeptide with preferential mitogenicactivity on cells of epithelial origin, said polypeptide comprisingamino acids 65-156 and 162-189 of FIG.
 7. 5. The DNA molecule of claim4, wherein the KGF polypeptide shows preferential mitogenic activity forBALB/MK epithelial cells, but not NIH/3T3 fibroblast cells.
 6. Anisolated cDNA molecule encoding a truncated keratinocyte growth factor(KGF) polypeptide which has preferential mitogenic activity for cells ofepithelial origin, wherein said polypeptide is truncated within theregion encoding amino acids 32-78 of the sequence of FIG. II-1B.
 7. ThecDNA of claim 6, wherein said truncated KGF polypeptide showspreferential mitogenic activity for BALB/MK epithelial cells, but notNIH/3T3 fibroblast cells.
 8. A host cell stably transformed ortransfected with a DNA molecule according to any one of claims 4, 1, 2or 6, in a manner allowing the host cell to express KGF, wherein saidhost cell is selected from the group consisting of a procaryote andeucaryote.
 9. A biologically functional vector comprising a DNA moleculeaccording to any one of claims 4, 1, 2 or
 6. 10. A method of producing aKGF polypeptide having preferential mitogenic activity for cells ofepithelial origin, the method comprising:(a) cultivating underappropriate nutrient conditions a host cell stably transformed with aDNA molecule according to any one of claims 4, 1, 2, or 6 in a mannerenabling production of the encoded polypeptide, and (b) isolating thepolypeptide so produced.
 11. A method of producing human KGF comprisingculturing under appropriate nutrient conditions a host cell according toclaim 8 in a manner allowing the host cell to express KGF and isolatingthe KGF so produced.
 12. The host cell according to claim 8, selectedfrom the group consisting of a bacterial, insect, mammalian and a yeastcell.
 13. The bacterial host cell according to claim 12 which is E.coli.
 14. A host cell stably transformed or transfected with a vectoraccording to claim
 9. 15. The host cell according to claim 14 that isselected from the group consisting of a bacterial, insect, mammalian anda yeast cell.
 16. The host cell of claim 15, wherein said bacteria is E.coli.
 17. An isolated DNA molecule encoding a keratinocyte growth factor(KGF) polypeptide having preferential mitogenic activity on cells ofepithelial origin, wherein the KGP polypeptide comprises amino acids32-78 of FIG. II-1B or a portion thereof, fused to the coding sequenceof a member of the fibroblast growth factor (FGF) family that is notKGF, wherein said coding sequence corresponds to amino acids 79-194 ofFIG. II-1B.
 18. A host cell containing the DNA of claim
 17. 19. The DNAmolecule of claim 17, wherein said KGF polypeptide shows preferentialmitogenic activity for BALB/MK epithelial cells, but not NIH/3T3fibroblast cells.
 20. A biologically functional vector comprising a DNAmolecule of claim 17, wherein said vector is a viral or plasmid vector.21. A host cell stably transformed with the vector of claim
 20. 22. Thehost cell according to claim 21, which is selected from the groupconsisting of a bacterial, insect, mammalian and a yeast cell.
 23. Amethod of producing a chimeric polypeptide comprised of a KGFpolypeptide having preferential mitogenic activity for cells ofepithelial origin wherein the KGF polypeptide comprises amino acids32-78 of FIG. II-1B or a portion thereof, fused to the coding sequenceof a member of the fibroblast growth factor (FGF) family that is notKGF, wherein said coding sequence corresponds to amino acids 79-194 ofFIG. II-1B, the method comprising:(a) cultivating under appropriatenutrient conditions a host cell stably transformed with a DNA moleculeaccording to claim 145, in a manner enabling production of the encodedpolypeptide, and (b) isolating the polypeptide so produced.
 24. Themethod of claim 23, wherein said chimeric polypeptide shows preferentialmitogenic activity for BALB/MK epithelial cells, but not NIH/3T3fibroblast cells.
 25. An isolated DNA molecule comprising a DNA sequencewhich is complementary to the DNA sequence of FIG. II-1B.
 26. Anisolated DNA molecule comprising a DNA sequence which is complementaryto the polypeptide coding region in FIG. II-1B.
 27. A DNA moleculeaccording to claim 4, wherein said DNA molecule further comprises a 5'ATG.
 28. The DNA molecule of claim 1, wherein said mammalian DNA ishuman.
 29. A DNA molecule according to claim 6, wherein said DNAmolecule further comprises a 5' ATG.